Electrical response of retinal ganglion cells in an N-methyl-N-nitrosourea-induced retinal degeneration porcine model

Retinal prosthesis is regarded as the treatment for vision restoration in the blind with retinal degeneration (RD) due to the loss of photoreceptors. A strategy for retinal prosthesis is to electrically activate surviving neurons. The retina’s response to electrical stimulation in a larger RD model has not been studied yet. Therefore, in this study, we investigated electrically evoked retinal responses in a previously validated N-methyl-N-nitrosourea (MNU)-induced porcine RD model. Electrically evoked responses were evaluated based on the number of retinal ganglion cell (RGC) spikes via multichannel recordings. Stimulation pulses were applied to degenerative and wild-type retinas with pulse modulation. Compared to wild-type retinas, degenerative retinas showed higher threshold values of pulse amplitude and pulse duration. The rate of increase in the number of RGC spikes relative to stimulus intensity was lower in degenerative retinas than in normal retinas. In severely degenerated retinas, few RGCs showed electrically evoked spikes. Our results suggest that the degenerative porcine retina requires a higher charge than the normal porcine retina. In the early stage of RD, it is easier to induce RGC spikes through electrical stimulation using retinal prosthesis; however, when the degeneration is severe, there may be difficulty recovering patient vision.

, further research is required in this regard. Because their results were analyzed based only on the direct RGC responses with a short latency (less than 20 ms) without considering indirect responses of RGCs, investigating the indirect responses of RGCs through retinal synapses among different mammals is indeed crucial. More importantly, it is necessary to optimize parameters for electrical stimulation of RGCs using models with larger mammals that are evolutionarily closer to humans. We chose the porcine model because of several similarities in the eyes of pigs and humans, not only in the size of the eyeball but also in its composition and the shape of the retina 27 . Previously, we developed an N-methyl-N-nitrosourea (MNU)-induced porcine RD model, and showed that the porcine model is a good animal model for patients with RP 28 . In this study, we investigated the correlation between the severity of RD and electrically elicited RGC responses using the same porcine model.

Results
Classifying in-vitro RD severity. In our previous study, we reported the reduced light responsiveness in MNU-induced porcine RD model using the full-field electroretinogram (ffERG) recording 28 , wherein ERG recording represented the overall average responses of photoreceptors (a-wave) and BCs (b-wave). In this study, we focused on the responses of individual RGCs using an in-vitro multi-electrode array (MEA) recording setup to identify changes in light responsiveness in local areas (2 mm × 2 mm) of the retinal tissue, not the whole retina (Fig. 1A).  Supplementary Fig. S1). As a control group, the non-MNU-treated (normal) group showed 66.1 ± 2.2% (mean ± SEM) LS-responsiveness (n = 9 retinal patches) ( Fig. 2A). We classified MNU-treated retinal patches (n = 15) into two groups, non-severe and severe RD, based on the existence and non-existence of LSresponsive RGCs, respectively. If the tested retinal patch did not respond to LS, we classified the retinal patch as a severe RD group. The other retinal patches were classified as the non-severe RD group. The average percentage of LS-responsiveness in the non-severe RD group was 48.4 ± 2.2% (n = 7 retinal patches).
Responsiveness to electrical stimulation of RGCs according to the degree of RD severity. We previously found that even after the photoreceptor layer degenerated, RGCs remained intact 28 . Therefore, in the current study, we speculated that RGCs would be differentially responsive to electrical stimuli (ES) with varied current pulses (duration: 0.5, 1, and 2 ms; amplitude: 10, 15, 20, 25, and 30 μA). Figure 2B shows the percentage of ES-responsive RGCs in the three RD groups at different severity of degeneration at the strongest level of stimulus (2 ms pulse duration and 30 μA pulse amplitude). No significant differences were observed in the percentage of ES-responsive RGCs between the normal and non-severe RD groups (56.1 ± 8.7% and 59.3 ± 6.6%, respectively). However, only about half of the average percentage of ES-responsive RGCs (27.8 ± 8.9%) were seen in the severe RD group.
MNU-treated RD retina is well modulated by the current stimulation protocol, similar to the normal retina. In our previous study, we observed that RGCs in general do not respond proportionally to changes in the strength of electrical stimulation in mice 29 . We also found such non-proportional RGC responses in pigs to various levels of pulse amplitude modulation (PAM) and pulse duration modulation (PDM). Therefore, in this study, we selected only well-modulated RGCs that showed a proportional response curve drawn from the levels of RGC responses to the various combinations of electrical stimulation with different durations (0.5, 1, and 2 ms) and amplitudes (10,15,20,25, and 30 μA). The ES-responsive RGCs of each group are described in Fig. 2B. Briefly, only 17.36% (75/432), 18.41% (58/315), and 6.90% (12/174) of RGCs were well-modulated throughout all three PAM and five PDM protocols in the normal, non-severe, and severe RD groups, respectively (Fig. 3). www.nature.com/scientificreports/ Figure 4 shows the curves of the average RGC responses of well-modulated RGCs in three different severities of the RD groups. In the normal group, the RGC responses tended to increase proportionally with an increase in pulse amplitude and duration. However, MNU-treated groups showed saturated RGC responses even at the stimulation level that induced a proportional increase in the normal group. In the non-severe RD group, the RGC response curve started to saturate from a pulse amplitude of approximately 20 μA with a fixed pulse duration of 1 ms. In addition, the RGC responses were rarely modulated by a 2-ms duration fixed pulse. In the severe RD group, the RGC responses were rarely modulated by fixed 1-or 2-ms duration pulses. Similar to the results of the PAM experiment, the RGC responses tended to increase proportionally with an increase in pulse duration in the normal and non-severe RD groups. However, in the severe RD group, RGC responses were rarely modulated by all the PDM protocols. www.nature.com/scientificreports/ Thresholds of pulse amplitude and pulse duration differ according to the degree of RD severity. Next, we obtained thresholds for pulse amplitude and pulse duration that evoked spikes in well-modulated RGCs (Fig. 5). In all PAM protocols (0.5, 1, and 2 ms fixed pulse), the RD groups in all three degrees of severity showed decreased thresholds of pulse amplitude with an increment of fixed pulse duration. In addition, in all PDM stimulations, all three RD groups showed decreases in thresholds for pulse durations with an increment of fixed pulse amplitude. The obtained threshold values and threshold charge densities are described in Table 1.

Strength-duration (SD) curve from well-modulated RGCs.
We constructed a strength-duration curve of well-modulated RGCs. The SD curve was fitted with a hyperbolic function (I = R(1 + C/D), I = amplitude, D = duration, R = rheobase, and C = chronaxie; Fig. 6A). Rheobase and chronaxie were extracted from the fitted curves. The average SD curves seemed to shift upward and to the right as the severity of RD increased; however, there were no noticeable differences in rheobase and chronaxie in the three groups (Fig. 6B). The average values of rheobase were 3.37 ± 0.36, 3.94 ± 0.40, and 4.80 ± 1.05 μA (mean ± SEM) in the normal, nonsevere RD, and severe RD groups, respectively. The average values of chronaxie were 4.21 ± 0.95, 6.32 ± 2.11, and 6.84 ± 4.48 ms (mean ± SEM) in the normal, non-severe RD, and severe RD groups, respectively.

Discussion
In this study, we successfully demonstrated that an MNU-induced RD pig model can be used to optimize parameters for electrical stimulation to further help develop retinal prosthetics. We previously showed that our new RD model could be successfully induced with MNU in a larger animal 28 . In that study, the results were evaluated by multimodal examinations, including spectral-domain optical coherence tomography (OCT) imaging, ffERG recording, and immunohistochemistry. The MNU-induced RD pig model up to moderate RD mimicked human RP patients well in terms of rod dominant ERG abnormality which shows a decrease of ERG amplitude and delayed implicit time of a and b waves in scotopic condition. However, in the severe RD model, there is a mismatch between the animal model and human RP patients, in terms of RGC death in the central retina. This unexpected RGC death observed in the animal model might be coming from the local heterogeneities in RD expression in the porcine model. Since our previous evaluations were mostly www.nature.com/scientificreports/ based on the morphological evidence observed in the outer layer of whole retinas and the global response of the retina via ffERG recording, we need to look into the electrophysiological activities of RGCs that are essential for restoring vision in patients with RD using retinal prosthetics. Moreover, the in-vitro MEA recording with isolated retina is easy to handle in comparison with the in-vivo recording as EEP signal with the living animal. This benefit of the in-vitro recording facilitates the repetition of the experiment. By repeating experiments, we can generate more reliable data, which is one of the strengths  www.nature.com/scientificreports/ of in-vitro study over in-vivo study. Thus, we tried to find the electrical stimulus parameters such as threshold charge density using the in-vitro MEA recording setup. The in-vitro study detects the RGC response at the retina to electrical stimulation while in-vivo EEP recording detects the visual percepts at the visual cortex. With this difference, the current threshold for RGC spikes or EEP signal should be different.
In this in-vitro study, the threshold current of 8 μA and the threshold charge of 4 nC in wild-type pig retinas are derived. On the other hand, Laube et al. showed the threshold current of 50 μA and the threshold charge of 10 nC for in-vivo EEP recordings in minipigs under epiretinal stimulation 30 . However, our threshold charge density and Laube's are compatible, respectively 0.58 mC/cm 2 and 0.26 mC/cm 2 . To our best knowledge, no report for EEP recording can be found using the porcine RD model. Therefore, no comparison is possible between in-vitro recording and in-vivo recording in the porcine RD model. According to literature analysis based on various animal models except for the porcine RD model, there is no difference between threshold charge density with in-vitro recording in the retina and in-vivo recording in the cortex 19 . Thus, finding electrical stimulation parameters such as threshold charge density using an in-vitro setup is meaningful and provides a guideline for in-vivo studies or clinical trials.
Clinically used retinal implants for decades, such as the Argus II and Alpha IMS, are known to stimulate RGCs locally, covering only the macular area of the retina. Because the sizes of the electrode array in the Argus II and Alpha IMS (3.8 mm × 6.7 mm and 3 mm × 3 mm, respectively) 8,31,32 and that of MEA used in this study (2 mm × 2 mm) are relatively compatible, our findings can be especially useful to estimate RGC responses to retinal prosthetic devices that will be implanted in the retinas of patients with RD.
We observed that the MNU-induced RD porcine model followed progressive photoreceptor death, similar to that observed in the genetically induced RD model 33 . Although the degeneration process observed in the www.nature.com/scientificreports/ MNU-treated model was similar to that of genetically induced ones, there were differences in the distribution of degeneration in the retina. Unlike genetically induced RD, which causes global homogeneous expression of RD throughout the retina, MNU-treated RD exhibited local heterogeneities in RD expression even when MNU was spread evenly throughout the retina 28,34 .
Because of this inconsistency in the expression of RD, the retinal patches that we recorded showed varied severity in degeneration, which may have been neglected in our previous study that used the average of amplitude changes in the ffERG recording 28 . Therefore, to assess the severity of RD more accurately (Fig. 2), we classified recorded neurons into two different RD groups based on the presence of LS-responsive RGCs as severe and nonsevere RD groups. We found that the number of ES-responsive RGCs in the non-severe RD group was similar to that in the normal group, whereas the number of ES-responsive RGCs in the severe RD group decreased to about half of the numbers in the non-severe RD group.
Further, the RGC responses evoked by electrical stimulation can be categorized into two groups: direct and indirect. The responses elicited by the direct excitation of electrical stimulation on RGCs occur within 10 ms after the stimulus, whereas the indirect responses are observed from 10 to 100 ms after the stimulus onset, presumably because of the time taken during synaptic transmission from photoreceptors or BCs to RGCs 23,35 . As mentioned in the "Methods" section, since we only investigated ES-responsiveness of indirect response, the reason for the reduced number of ES-responsive RGCs in the severe RD group may be the functional loss of BCs. Histological evidence from our previous study showing destruction in the inner nuclear layer (INL) in the retinas of severe MNU-treated RD pigs supports this hypothesis 28 .
Furthermore, we observed that the ES-responsive RGCs in MNU-treated retinas showed less ES-responsiveness than normal retinas of pigs (Figs. 4, 5). Specifically, the ES-responsiveness of MNU-treated retinas was saturated by relatively weaker electrical stimulation than that of normal retinas. This means that there is a limit to evoking RGC responses regardless of the intensity of electrical stimulation. In other words, stronger electrical stimulation will not be effective in RD retinas once it passes the point where the RGC responses start to saturate.
We were able to stimulate RGCs with following threshold charge density (normal: 0.55 ± 0.07 mC cm −2 phase −1 ; non-severe group: average charge density = 0.78 ± 0.08 mC cm −2 phase −1 , calculated from the data in Table 1). In comparison with a lower animal model, the RGCs in the normal and RD porcine retina required higher threshold charge density (for instance, WT mice required 0.35 ± 0.05 mC cm −2 phase −1 and rd1 mice required 0.41 ± 0.08 mC cm −2 phase −1 , calculated from our previous study 11 ). However, because the amount of electrical charge density measured in this study was less than the safety limits found in earlier studies 19,21 , the degenerated retina of the porcine model could respond with a relatively safer range of electrical stimulation. This is an important finding that should be considered when developing neural prosthetic devices.
The overall thresholds for amplitude and duration to evoke RGC responses in the MNU-treated retinas were higher than those in normal retinas of pigs (Table 1). These results have been observed in other animal models of RD. In our previous study, we reported that the RGCs in the completely degenerated retinas of rd1 mice required higher intensities of electrical stimulation to elicit responses than the wild-type RGCs of C57BL/6J mice 11 . Moreover, phosphenes in human patients with RP are less induced by electrical stimulation than that in sighted human subjects 36,37 . Collectively, these results can help optimize parameters for electrical stimulation in patients with RD in the future.
Although it is difficult to identify the exact sources of the reduced ES-responsiveness in RD retinas observed in this study, the following plausible causes are worth considering that can eventually help overcome the technical obstacles that retinal prosthetic devices render: 1. Glia sealing: Gliosis by Müller glial cells appears in degenerated retinal networks after the total death of photoreceptors 38 . It is also known that Müller glial cells can seal both outer nuclear layers and ganglion cell layers. This enclosure of RGCs by glial cells has been also reported in all cases of genetically induced RD, MNU-induced RD animal models, and human patients with RP 28,34,[39][40][41] . This sealing effect on RGCs may have prevented the surviving neurons from being stimulated by electrical pulses in the degenerated retina in the present study. No significant differences were observed among the chronaxie and rheobase values in all three groups (Fig. 6). This finding implies that the electrical properties of RGCs, even in the severe RD groups, are well preserved regardless of the severities of RD, thereby maintaining electrical responsiveness. This resilience of RGCs makes them possible to be evoked by electrical stimulation using a retinal prosthesis. Sekirnjak et al. studied chronaxies and rheobase of normal RGCs in three different animals, namely monkeys, guinea pigs, and rats 19 . They reported similar patterns in the SD relationship regardless of the species. However, their averaged values of chronaxie and rheobase (338 ± 81 μs and 0.60 ± 0.11 μA) were much lower than those found in this study (4210 ± 950 μs and 3.37 ± 0.36 μA). This discrepancy may be due to the difference in origins of the RGC spikes. Sekirnjak et al. mainly investigated direct responses, whereas we focused on indirect responses. Several previous studies have reported that BCs indeed require a longer pulse duration than RGCs 14,42 .
In conclusion, our results suggest that the degenerative porcine retina requires a higher charge than the normal porcine retina. In addition, in the early stage of RD, it is easy to induce RGC spikes through electrical www.nature.com/scientificreports/ stimulation using a retinal prosthesis; however, when the degeneration progresses to a severe degree, recovering the patient's vision will be relatively difficult.

Methods
Animals. All  . The subjects were euthanized immediately after enucleation of the eyeball. The retina was isolated and cut into patches of approximately 3 mm × 3 mm from a visual streak (Fig. 1A). The detailed procedures for the preparation have been described in previous studies 22,43 (Fig. 1B).
Data recording system. The RGC spikes were recorded using a 60-channel MEA. Briefly, the data acqui- Light stimulation. The light stimuli were generated by custom-made software, written in MATLAB with Psychtoolbox 44,45 and presented via a liquid-crystal display (LCD) projector (AJD-36; OMAX, Bucheon-si, Gyeonggi-do, South Korea). We applied white full-field illumination (Light ON; intensity: 10.15 μW/cm 2 ) for 2 s and black background light (Light OFF; intensity 0.38 μW/cm 2 ) for 2 s alternatively. The ON and OFF cycles were sequentially repeated 20 times (Fig. 1C). The abovementioned light intensity corresponding to photopic vision (10.15 μW/cm 2 ) was measured using a calibrated radiometer (ILT-5000; International Light Technologies, Peabody, MA, USA).
Electrical stimulation. Among the 59 electrodes of the pMEA, an electrode at the center was selected for stimulation, and the others were used for recording. Pulse trains were applied to the stimulation electrode using a stimulus generator in the MEA2100-60-headstage. The stimuli consisted of symmetric, cathodic, phase-first biphasic pulses. Trains of 50 identical pulses were applied every 1 s along with an increase in the pulse amplitude (10,15,20,25, and 30 μA) and pulse duration (500, 1000, and 2000 μs) (Fig. 1C).

Data analysis.
From the raw waveform of the retinal recording, the RGC spikes were isolated using a 100 Hz high-pass filter with the offline data analysis software of the abovementioned Multi Channel Suite package (Multi Channel Analyzer; Multichannel Systems GmbH, Reutlingen, Germany). The threshold for spike detection was set to four times the standard deviation of the background noise. The recording data were processed with a spike sorting software (Offline Sorter™; Plexon Inc., Dallas, TX, USA) to separate multiunit activities containing different spike waveforms into individual cell units using principal component analysis. Data analysis was performed using a commercial analysis software (NeuroExplorer ® ; Nex Technologies, Colorado Springs, CO, USA) and a custom-made MATLAB (MathWorks, Natick, MA, USA) code. The RGC responses evoked by electrical stimulation were defined as the relative changes in the number of RGC spikes after the stimulation onset. To quantify the spontaneous RGC firings, we calculated the average number of spikes before the stimulation onset in the 20 bins (bin = 100 ms). Next, we calculated the average number of spikes in 20 trials of electrical stimulation within the post-stimulus period from 10 to 100 ms. We disregarded the signals from 0 to 10 ms to remove the stimulus artifact and direct RGC responses. The threshold